PROJECT SUMMARY Many cancers harbor translocations, amplifications, or activating mutations of the c-Myc proto- oncogene. Myc regulates many programs in the cell including growth and proliferation, and apoptosis as well, predominantly through the activation of p53. However, Myc deregulation and p53 inactivation frequently co-occur in B-cell lymphomas such as Burkitt's lymphoma (BL); thus, chemotherapies for patients are not always curative. Studies have shown that very high levels of oncogenic Myc are required to engage apoptosis, thus one strategy to improve current therapies would be to transiently increase Myc expression and stability. GSK3? regulates Myc protein stability by phosphorylating Myc at the Thr58 residue, marking it for ubiquitylation and degradation. GSK3? can be targeted pharmacologically with specific inhibitors, making it an attractive target to modulate Myc levels. Preliminary data show that transient up-regulation of Myc by inhibiting GSK3? with CHIR99021 enhances apoptosis in B-cell lymphomas when combined with chemotherapeutic drugs and suggest that the extrinsic apoptotic pathway is important for this enhanced apoptosis. Extrinsic apoptosis is activated by binding of ligands (FasL, TNF, TRAIL) to death receptors on the cell surface (Fas, TNFR1, DR4, DR5). The goal of this proposal will be to determine the mechanism by which anti- GSK3? adjuvant therapy enhances apoptosis in B-cell lymphomas and to investigate both Myc- dependent and independent mechanisms. My first aim will be to examine the role of Myc and other GSK3? targets in anti-GSK3? adjuvant therapy. To assess the role of Myc, I will compare induction of apoptosis in BL cells with WT vs. mutant Thr58 Myc, where Myc cannot be stabilized upon GSK3? inhibition. I will investigate PTEN as another GSK3? target and validate the effect of GSK3? inhibition on PTEN protein level, stability, Thr366 phosphorylation (the site targeted by GSK3?) and activity. I will then compare the response of BL cells engineered with WT vs. Thr366 mutant PTEN to anti- GSK3? adjuvant therapy. My second aim will investigate the contribution of extrinsic apoptosis by using CRISPR/Cas9 to delete death receptors on BL cells complemented by modulation of FLIP-long expression, the negative regulator of the extrinsic pathway; I will test the response of these modified cells to anti-GSK3? adjuvant therapy. Finally I will investigate the role of the extrinsic pathway in vivo by comparing the response of subcutaneous xenografts of BL cells with intact vs. perturbed extrinsic apoptotic signaling to anti-GSK3? adjuvant therapy as well as GSK3? inhibition + the extrinsic ligand TRAIL. These proposed experiments will provide rationale and mechanism for GSK3? inhibition as adjuvant therapy for Myc-driven B-cell lymphomas.